DNA repair and recombination are required by organisms to prevent the accumulation of mutations and to maintain the integrity of chromosomally encoded genetic information. Compromised genetic information is often associated with cell cycle arrest, programmed cell death, or cell senescence. Compromised genetic information has also been linked to cell cycle aberrations that are often associated with uncontrolled cell growth and possibly tumor formation.
Enzymes capable of repairing double-strand breaks (DSB) in DNA perform an essential function found in nearly all living cells. DSB repair may occur in conjunction with general DNA repair, or in a species-specific manner such as the mating-type switch in Saccharomyces cerevisiae and V(D)J (Variable [Diversity] Joining) recombination in mammals (reviewed by Friedberg et al., 1991; Haber, 1992, Trends Genet. 8:446-452; Roth et al., 1995, Current Biology 5:496-499). In bacteria and yeast cells, DSB are predominately repaired by a homologous recombination pathway (Mortimer, 1958, Radiat. Res. 9:312-316; Krasin and Hutchinson, 1977, J. Mol. Biol. 116:81-98). However, in mammalian cells, DSB may be repaired by either a homologous or a nonhomologous recombination pathway (Rouet et al., 1994, Mol. Cell. Bio. 14:8096-8106; Choulika et al., 1995, Mol. Cell. Bio. 15:1968-1973).
In the budding yeast, S. cerevisiae, repair of DSB occurs through a homologous recombination pathway that depends on the RAD52 epistasis group (Rad50-Rad57), which was identified in cells sensitive to ionizing radiation. Some members of this group were shown to be important for recombinational repair during mitotic and meiotic recombination (for reviews see Game, 1983, Radiation-sensitive mutants and DNA repair in yeast. p. 109-137. In: "Yeast genetics: fundamental and applied aspects." J. F. T. Spencer, D. Spencer, and A. R. W. Smith (eds.), Springler-Verlag, New York; Petes et al., 1991, Recombination in yeast, In: The Molecular and Cellular Biology of the Yeast Saccharomyces, pp. 407-521, J. R. Broach, J. R. Pringle, and E. W. Jones (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Among the members of the RAD52 epistasis group, ScRad51 is interesting because of its similarity to the Escherichia coli recombination protein, RecA (Shinohara et al., 1992, Cell 69:457-470; Aboussekhra et al. 1992, Mol. Cell. Biol. 12:3224-3234; Basile et al., 1992, Mol. Cell. Biol. 12:3235-3246). Both enzymes share approximately 30 percent homology over a region of about 220 amino acids and polymerize on double-stranded and single-stranded DNA (dsDNA, ssDNA), showing a nearly identical helical filament (Ogawa et al., 1993, Science 259:1896-1899; Sung and Robberson, 1995, Cell 82:453-461). ScRad51 and RecA catalyze an ATP-dependent strand exchange between homologous DNA molecules (Sung, 1994, Science 265:1241-1243; Sung and Robberson, 1995; for review see Radding, 1991, J. Biol. Chem. 266:5355-5358).
ScRad51 repairs DSB by recombination, and DSB accumulate at recombination hot spots in cells that lack ScRad51 during meiosis (Shinohara et al., 1992). ScRad51 and another RecA homologue, DMC1, colocalized to meiotic nuclei (Bishop, 1994, Cell 79:1081-1092) and promoted meiotic chromosome-synapsis (Rockmill et al., 1995, Genes & Devel. 9:2684-2695). Therefore, ScRad51 may mediate meiotic recombination by binding to single strands generated at DSB which could lead to pairing and strand exchange during meiosis as suggested by Sung and Robberson, 1995.
RecA/ScRad51 homologues have been discovered in a wide range of organisms including the fission yeast Schizosaccharomyces pombe (Shinohara et al., 1993, Nature Genet. 4:239-243; Muris et al., 1993, Nucleic Acids Res. 21:4586-4591; Jang et al., 1994, Gene 142:207-211), lilies (Terasawa et al., 1995, Genes & Dev. 9:925-934), chicken (Bezzubova et al., 1993, Nucl. Acids Res. 21:1577-1580), mouse (Shinohara et al., 1993; Morita et al., 1993, Proc. Natl. Acad. Sci., U.S.A., 90:6577-6580) and human (Shinohara et al., 1993; Yoshimura et al., 1993, Nucleic Acids Res. 21:1665). RecA/ScRad51 homologues also appear to be involved in DNA repair, and recombination as based on the following indirect evidence: 1) Conserved RecA homology--MmRad51 is 83% homologous and 69% identical to ScRad51, and 51% homologous and 28% identical to RecA (Shinohara et al., Morita et al., 1993). Shared homology between mammalian and yeast Rad51 suggest conserved function due to the remarkable similarity between other mammalian and yeast repair pathways (reviewed by Cleaver, 1994, Cell 76:1-4); 2) Expression pattern: MmRAD51 is highly expressed in tissues involved in meiotic recombination (testis and ovary; Shinohara et al., 1993; Morita et al., 1993) and expression of the S. pombe homologue, SpRAD51 (also called rhp51+), increased after cells were treated with methyl methanesulfonate suggesting a role in DNA repair (Jang et al., 1994); 3) Protein cellular localization: Mouse, chicken, and lily Rad51 localized as discrete foci on meiotic chromosomes at varying concentrations during prophase 1, possibly on the lateral elements and recombination nodules, suggesting a role in the repair of DSB during meiotic recombination (Haaf et al., 1995, Proc. Natl. Acad. Sci., U.S.A. 92:2298-2302; Ashley et al., 1995, Chromosoma 104: 19-28; Terasawa et al., 1995). Human Rad51, HsRad51, located to the nucleus with increasing concentration after exposure to DNA damaging agents suggesting a repair function (Haaf et al., 1995); 4) Filament formation on DNA: HsRad51 bound to ssDNA demonstrating a potential for strand exchange (Benson et al., 1994, EMBO 13:5764-5771). The present invention has provided the first direct evidence of the function of mammalian Rad51 and provides insight into recombinational repair in animal cells.